1. Field of the Invention
The present invention relates to the field of casein materials, casein compositions, and methods of manufacturing casein in an edible form that is also resistant to dissolving in neutral or basic aqueous systems.
2. Background of the Art
Casein comprises a group of proteins that forms about 80 percent of the total proteins in cow""s milk. It solidifies when milk is made slightly acidic and is the chief ingredient in cheese. Casein is used as a food supplement, an adhesive, and a finishing material for paper and textiles. It is also used in water paints. Consumer demands for both higher quality and longer shelf-life foods have stimulated edible film research. The environmental movement has promoted increased concern about reducing disposable packaging amounts and increasing packaging recyclability, farther contributing to the recent surge in edible coating and film research. Edible films and coatings are capable of offering solutions to these concerns by regulating the mass transfer of water, oxygen, carbon dioxide, lipid, flavor, and aroma movement in food systems. Edible coatings function by direct adherence to food products; whereas, edible films act as stand-alone sheets of material used as wrappings, low moisture baked products, and intermediate and high moisture foods all exhibit potential for improvement through the use of edible coatings and films. Dried foods (e.g., dried vegetables and dried meats) and low moisture baked products (e.g., crackers, cookies and cereals) are particularly susceptible to moisture uptake from the atmosphere. Low moisture baked foods are also susceptible to moisture uptake from moist fillings and toppings. Such changes can result in loss of sensory acceptability of the food product, as well as a reduced shelf-life. Many dried and baked products are also susceptible to oxidation, lipid migration and volatile flavor loss.
Intermediate moisture foods, such as raisins and dates, often become unacceptable due to moisture loss over time. Moisture loss is particularly problematic when the moisture transfers into lower moisture components of a food system, For example, raisins can lose moisture to the bran in raisin bran. Nut meats, another intermediate moisture food, are susceptible to lipid oxidation resulting in the development of off flavors. High moisture food components typically lose moisture to lower moisture components. One classical example of this phenomenon occurs when pizza sauce moisture migrates into the crust during storage, resulting in a soggy crust. Oxidation and flavor loss are also problematic to high moisture food systems. The respiration rates of whole fruits and vegetables often dictate their shelf lives. Minimally processed fruits and vegetables are often subject to unacceptable levels of oxidative browning. Individual food products within the broad food categories discussed above require different barrier properties in order to optimize product quality and shelf-life. Edible films and coatings are capable of solving the barrier problems of these and a variety of other food systems. See, Kester, et al., Food Technol. 40:47-59 (1986) and Krochta, in Advances in Food Engineering, CRC Press, Inc., Boca Raton, Fla. Singh and Wirakartakusumab (Eds.) p. 517-538 (1992).
Edible films and coatings based on water-soluble proteins are typically water-soluble themselves and exhibit excellent oxygen, lipid and flavor barrier properties; however, they are poor moisture barriers. Additionally, proteins act as a cohesive, structural matrix in multicomponent systems to provide films and coatings having good mechanical properties. Lipids, on the other hand, act as good moisture barriers, but poor gas, lipid, and flavor barriers. By combining proteins and lipids in emulsion or bilayer barriers, the advantages of each component can be exploited to form an improved film system. Plasticizer addition improves film mechanical properties. It would be desirable to be able to provide water-insoluble protein films, even if they do not necessarily provide oxygen barriers.
The harvesting of casein from milk utilizing either acid or an enzyme precipitation while efficient for recovering the casein protein from the milk, does not recover any whey protein. After acid or enzyme precipitation of casein from milk, normally the whey fraction is discarded. This thus constitutes a waste of some of the protein content of the milk, even though the utilization of the whey has improved over the years. It has been suggested as, for instance, Phillips, et al. U.S. Pat. No. 4,218,490, to harvest the whey protein content of milk utilizing ion exchange resins.
U.S. Pat. No. 4,545,933 entitled Hydrolyzed Protein Composition and Process Utilized in Preparation Thereof describes a process for hydrolyzing casein protein utilizing caustic solutions of sodium or potassium hydroxide. The hydrolyzed protein produced by the process of this patent has certain unique properties which are useful in the preparation of certain processed food products. The starting materials suggested for the process of U.S. Pat. No. 4,545,933 is acid precipitated casein, that is casein which contains no whey protein.
The properties of composite bilayer films and coatings have been studied in the past. Cohesive bilayer films and coatings are often difficult to form and delamination may occur over time. Furthermore, bilayer film and coating formation often requires the use of solvents or high temperatures, making production more costly and less safe than aqueous emulsion film production. Protein-lipid emulsion film and coating systems can be formed from aqueous solutions and applied to foods at room temperature.
Water-insoluble edible films and coatings offer numerous advantages over water-soluble edible films and coatings for many food product applications. Increasing levels of covalent crosslinking in water-insoluble edible films and coatings result in better barriers to water, although not necessarily barriers to oxygen, carbon dioxide, lipids, flavors and aromas in food systems. Film mechanical properties are also improved. Many foods, such as fruits and vegetables, are exposed to water during shipping and handling. In these cases, water-insoluble films and coatings remain intact; whereas, water-soluble films and coatings dissolve and lose their barrier and mechanical properties. Edible films in the form of wraps, such as sandwich bags, also require water-insolubility.
Prior to this invention, water-soluble, protein-based edible films and coatings have been formed from aqueous solutions of proteins (Gennadios, et al., in Edible Coatings and Films to Improve Food Quality, Technomic Publishing Co., Lancaster, Pa., Krochta, Baldwin and Nisperos-Carriedo (Eds.), Chapter 9, (1994)); however, a means to produce water-insoluble films and coatings from aqueous solutions with improved barrier properties had not been discovered. Carmelization and/or Maillard browning reactions had been exploited for the formation of improved protein-based oxygen barrier coatings for fruits and vegetables (Musher, U.S. Pat. No. 2,282,801). Protein thiol-disulfide interchange and free thiol oxidation reactions had been studied previously (see Donovan, et al., J. Food Sci. and Technol. 11:87-100 (1987) and Shimada, et al., J. Agric. Food Chem. 37:161-168 (1989)). However, the use of these reactions for the formation of new and improved edible barriers had not been explored. Edible moisture barrier coatings had been formed out of protein-based aqueous emulsions (see, Adams, et al., EP 0 465 801 Al and Ukai, et al., U.S. Pat. No. 3,997,674). However, methods for the formation of water-insoluble protein-based films and coatings had not been discovered.
Others have studied the interactions between proteins and lipids at interfaces in emulsions and colloidal systems. See, Barford, et al., in Food Proteins, American Oil Chemists Society, Kinsella and Soucie, eds., (1989) and Le Meste, et al., in Interactions of Food Proteins, American Chemical Society, Washington, D.C., Parris and Barford, eds., (1991). However, regulation of mass transfer as a function of lipid particle size and distribution in films has not been explored.
What is needed is a method for preparing water-insoluble protein-based edible films and coatings from casein that exhibit improved water-solubility resistance, and barrier and mechanical properties.
Industrial application of casein, which is the major component of milk proteins, has been studied in many fields. Although some of these studies are actually practiced in industries, the amount of casein used in these applications is limited. The reason is that casein is difficult to be molded, extruded, or worked, and its physical properties are limited by its water-solubility. These physical limitations are in part a result of the fact that this protein easily forms a stable micelle structure due to its macromolecular surface activity.
Casein protein is a phosphoprotein possessing a macromolecular surface activity which enables the protein to form a micelle structure. The micelle structure renders casein stable in milk. When separated from milk, casein will form globular micelles if an alkaline earth metal, such as calcium or magnesium, is present. Such globular micelles are difficult to disperse in a medium and difficult to mold.
There have been several proposals for dispersing casein, however, none brought about good results. For example, Japanese Patent Laid-open (kokai) No. 138145/1987 discloses a method of dissolving caseinate in an ethanol aqueous solution and making films by fluid spreading (which might be a casting method). However, caseinate becomes too hard when heated in a drying step, preventing it from being formed into fibers or films. This method therefore has not been industrially successful. Journal of Japan Agrichemical Society, 61, 1087-1092 (1987) proposes a method of dissolving or dispersing casein molecules by breaking down the micelle structure in a casein solution. The method involves treating the casein solution with a chelating resin, thereby removing the metals, e.g. calcium, from the casein solution. The report states that after the treatment, the casein molecules form sub-micelles, but does not describe how the sub-micelles can be used.
Casein protein is a water-soluble macromolecular surfactant consisting of hydrophobic protein and hydrophilic phosphoric acid groups, the latter groups rendering the protein more soluble in acid solutions. The phosphoric acid groups are bonded to counterions, i.e. metals such as calcium and magnesium, which induce the protein to form globular micelles. This configuration makes casein molecules difficult to orient in the longitudinal direction necessary to obtain an acceptable fiber. Obtaining fibers and other molded articles with acceptable mechanical properties from casein, therefore, has not been successful.
The properties and structure of the casein molecule include characterizations as millions of particles/cc with an average path of 0.37 micrometers between particles. The Molecular Weight varies from 10 to 3280 million, and the shape of the molecule is likely spherical. The Composition Size of micelles depends on initial s:k-casein (sigma/kappa) ratio, absolute protein concentration and Ca++ concentration. If micelles are removed by ultracentrifugation, the sediment is a clear gel. When dispersed in water, get an opaque colloidal suspension. It is highly hydrated, with about 2.5 grams water/gm. protein. All casein subunits are accessible to high molecular weight reagents and the association of subunits is through noncovalent bonds.
The principal solid constituent of milk is casein, a protein. When milk is allowed to stand in a warm place, it sours, and the casein is precipitated by the action of lactic acid bacteria. The thick precipitate, or curd, is separated from the thin, watery residue known as whey. Today curd is usually prepared with rennet, which acts to speed the separation process. The next steps in the making of cheese are salting (for flavor and eventually to aid in curing) and pressing (to shape the cheese and eliminate more whey). The curd is then ready for curing and is stored under temperature- and humidity-controlled conditions for varying lengths of time. In general, the longer the curing or aging process, the more pronounced the flavor of the finished product. During curing, gases are formed within the cheese, and in some types they are unable to escape; this produces the holes characteristic of some cheeses. To aid the curing process, harmless blue-mold spores are introduced into the blue-veined cheeses (Roquefort or blue cheese), and white-mold spores are sprayed on the surface of such cheeses as Brie and Camembert. This produces a rind, which may be eaten. Cheese casein is not a preferred source of casein in the practice of the present invention.
Casein may be derived from any original source and may be prepared by conventional methods can be used as the raw material in the present invention without any specific limitations. Casein is the principal protein in milk (whether, fresh cow milk, fresh goat milk or non-fat dried milk, ultrafiltered milk, other available milk forms and sources, and exists in milk as a colloidal aggregate of protein together with phosphorus and calcium. Other metal ions, such as magnesium, also may be present. Casein most useful in the present invention may be precipitated from milk by the addition of CO2 to cause precipitation.
In an optional initial series of steps in the present method, the casein may be treated to remove some of the calcium and any other forms of metals which may be present, although some calcium must remain to provide some structure to the film. This may be accomplished by any method effective for selectively removing metal ions, such as, for example, ion-exchange or chelation. A preferred method of removing the metals comprises contacting an aqueous solution or dispersion of casein with a chelating agent. Any chelating agent may be used in the method, however resins having fixed chelating functional groups are preferred. Chelating resins having iminodiacetic acid groups as functional groups are most preferably used as the chelating resin. A method for removing metal ions from casein using a chelate-functional resin which is described in said report of Journal of Japan Agrichemical Society, supra. Preferably, the carboxyl terminals of the iminodiacetic acid groups in the resin comprise hydrogen ions (H+). H-type resins are preferred because in order to ensure complete removal of metal ions from the casein, therefore the absence of alkali metals such as sodium at the functional group terminal is imperative. An iminodiacetic acid functional resin which is useful in the present invention, for example, is Uniselex.(trademark). UR30 (trademark, manufactured by Unitica Co., Ltd.). This step is carried out by contacting an aqueous casein solution with the chelating resin under conditions appropriate to remove some all of the metal ions from the casein solution, thereby forming sub-micellular casein. Sub-micellular casein thus obtained may be dried by a conventional method, if desired.
According to the present invention casein, which has been heretofore difficult to form into molded articles such as fibers, films, or the like, can be easily molded and manufactured into regeneration films, and it is readily predictable that fibers and other articles made of natural casein proteins may be made from the materials of the present invention. Casein fibers can be woven into cloth or sheets which are useful for various applications. In addition, because the raw material (casein) is naturally found in milk, articles made according to the invention that may be used as food materials, such as edible fibers. Casein is a naturally occurring material which is biodegradable, therefore, articles made from casein fibers or films contribute to global environment conservation. Casein films, non-woven fabrics of casein fibers or woven fibers and yarns, for example, could be used to make biodegradable packaging materials.
In an attempt to improve the structural stability of articles made from starch-based compositions, other ingredients have been included in the formulations. For example, compositions have been developed that include starch in combination with a water-insoluble synthetic polymers. Unmodified starches have also been combined with protein to provide moldable, biodegradable thermoplastic compositions. For example, Nakatsuka et al. (U.S. Pat. No. 4,076,846; issued Feb. 28, 1978) discloses an edible binary protein-starch molding composition containing a salt of a natural protein (i.e., casein), an unmodified, high amylose starch material, an edible plasticizer (i.e., sorbitol), and a lubricant (i.e., a fatty acid polyol ester), and having a final water content of about 10-40%. The composition is molded, for example, by extrusion through a die, into an article having a water content of about 5-30 wt-%. A disadvantage of these starch-based plastics is that the molded articles made from such compositions have a high tendency to absorb water, which causes the articles to lose mechanical strength and to disintegrate quickly.
U.S. Pat. No. 5,543,164 describes a method of forming an edible, protein-based water-insoluble film by treating a solution of the protein to effect disulfide formation and a denatured protein solution, then forming the denatured protein solution into a film. The denaturing is effected by causing a thiol-disulfide exchange by heat treatment and/or chemical reaction, e.g., heating between 70 and 95 degrees Celsius for up to three hours to initiate disulfide crosslinking reactions. These relatively high temperatures are essential for enabling the crosslinking to occur. The casein referred to here is probably calcium or sodium caseinate, usually manufactured by adding calcium or sodium hydroxide to acid casein (e.g., manufactured by the HCl process) and heated to at least about 75 degrees C. This type of calcium caseinate will have approximately the same molar proportions of casein with natural calcium therein, but the calcium in the calcium caseinate does not hold the micelles together as occurs with the natural calcium. The use of the vacuum is to assist in the removal of air bubbles that tend to get trapped in film after mixing, pouring or other mechanical procedures.
Conventional concentrating processes depend upon direct chemical treatment of the source vegetable matter to concentrate the protein. For example, raw soy products such as soy meal, soy flakes, and soy flour are treated with acid (e.g., hydrochloric acid) to precipitate protein and separate the protein from whey, sugars, oils and proteins which will not precipitate. Some of the acid remains in the protein precipitate and must be removed by additional processing either specific or generic to removal of the acid residue. As the acid is undesirable from many standpoints of flavor, aesthetics and health, it is desirable that in at least some uses that substantially all of the acid (reduced to an acid level of less than 0.5% by weight) is removed. The processing necessary to do this may be sufficiently harsh as to reduce the value and content of the soy protein concentrate or soy protein isolate produced by the acid treatment process.
There are also many physical processes for producing protein-rich products from grains. U.S. Pat. No. 5,135,765, for example, describes a process for producing a protein-rich product from brewer""s spent grain containing germ, husks and a proteinaceous material. The process requires the use of high water content spent grain (e.g., at least about 65% water by weight), passing the wet spent grain through a mill to press and grind the solids, and then sieving the spent grain in water to produce an at least 50% by weight protein product. After formation of the first concentrate, the coarse fraction may be extracted with alkaline aqueous solution at elevated temperature to form an extract, and the extract is acidified to form a further concentrated protein rich precipitate.
It is well known that soy bean products may have undesirable taste components. These components are known to be reduced or removed by selection of unique varieties of soy beans for the original source, heating an intermediate soy bean product to reduce lipoxygenase, extraction with an aqueous solution, extraction with an alkali solution, extraction with a reducing agent (e.g., see U.S. Pat. No. 5,023,104), extraction with organic solvents (e.g., removal of chlorohydrins from hydrolyzed protein compositions in U.S. Statutory Registration No. H989) and extraction with high pressure or supercritical carbon dioxide (e.g., xe2x80x9cPreparation and Evaluation of Supercritical Carbon Dioxide Defatted Soybean Flakesxe2x80x9d A. C. Eldridge, et al., Journal of Food Science, Vol. 51, No. 3, 1986, pp. 584-587; xe2x80x9cOff-Flavor Removal from Soy-Protein Isolate by Using Liquid and Supercritical Carbon Dioxidexe2x80x9d JAOCS, Vol. 72, no. 10, 1995, pp. 1107-1115; xe2x80x9cEmulsifying Properties of Low-fat, Low-cholesterol Egg Yolk Prepared by Supercritical CO2 Extractionxe2x80x9d Journal of Food Science, Vol. 61, No. 1, 1996, pp. 19-23 and 43; and U.S. Pat. No. 4,493,854 shows extraction of oil from soy (e.g., flakes) by CO2 extraction, leaving extracted meal as a by-product. The purpose of the process is to improve the flavor of the soy products by removal of undesirable flavor materials in the soy product. The process in U.S. Pat. No. 4,493,854 produces an enhanced flavor oil by tempering the initial soy material with moisture and extracting oil from the tempered soy product, but the by-product of protein and other solids is not a significantly concentrated product and is not an isolate, as it would still contain the whey, sugars and other materials not extracted by the CO2.
Bovine milk contains about 3 to 4% protein. The casein component of the bovine milk protein constitutes about 80% of the total protein. The remaining protein is divided among certain whey proteins with the principal one being .beta.-lactoglobulin.
It was recognized in antiquity that the casein protein of bovine milk could be separated from the xe2x80x9cwheyxe2x80x9d fractions by in situ acidification of milk utilizing enzyme extracts or by the direct addition of acid to the milk. For the preparation of casein from milk, after skimming the cream off the top the milk is acidified either by the addition of acid or by an enzyme. Below about pH 4.7 the casein precipitates as xe2x80x9ccurdxe2x80x9d leaving a clear liquid, the xe2x80x9cwheyxe2x80x9d.
In order to improve the heat sealability of edible films and thereby overcome the above-described disadvantages, a number of methods have been proposed. They include the method of forming a film from an intimate blend of amylose, an alkali metal salt of casein, and a low-molecular-weight plasticizer (Japanese Patent Laid-Open No. 112533/""76); the method of dipping a collagen film in, or coating it with, a mixture of gelatin or glue and a plasticizer (Japanese Patent Laid-Open No. 11280/""77); the method of forming a film by laminating a polysaccharide with gum arabic, pullulan, starch or gelatin (Japanese Patent Laid-Open No. 76336/""85); and the method of incorporating a solid fat in an edible film (Japanese Patent Laid-Open No. 59855/""88).
However, the films formed from an intimate blend of amylose, an alkali metal salt of casein, and a low-molecular-weight plasticizer, the films formed by laminating a polysaccharide with gum arabic, pullulan or starch, and the edible films having a solid fat incorporated therein still fail to exhibit adequate heat sealability. The films formed by laminating a collagen film or a polysaccharide with gelatin show a marked improvement in heat-seal strength, but have the disadvantage that the presence of gelatin in the surface layer causes severe blocking of films and this makes it difficult to handle the films.
An edible, water-resistant composition that can be formed into shaped articles including film comprises a water-resistant solid composition of casein. The casein composition may be directly precipitated from a solution under high pressure treatment with carbon dioxide. The composition does not have to be crosslinked, but takes advantage of the natural water-insolubility of the protein backbone of the casein. The casein composition may be combined with edible or inert flexibilizers to improve film properties, and the film may be used to protect food products or food compositions, yet provide moisture protection.